A quantitative FRET approach to characterize protein-protein interactions in living cells

The ability of proteins to specifically interact with each other is a key feature in the regulation of biological processes. Knowledge about interaction partners and characterization of protein-protein interactions contribute to the understanding of proper protein function and cell physiology. In particular, Förster resonance energy transfer (FRET) is a suitable method to analyze interactions between proteins in living cells. During the progress of this thesis, a quantitative FRET approach was established that aims to evaluate binding curves for interaction partners. Moreover, the quantitative FRET approach was applied to study biological questions, including the investigation of putative interaction partners of the endolysosomal ion channel two-pore channel 2 (TPC2), the Kv7.2 potassium channel and the photoreceptor-specific transmembrane protein peripherin 2. The FRET approach described in manuscript I computes calibrated FRET efficiencies from fluorescent measurements using three-filter cubes and correlates the FRET efficiencies to the concentration of donor and acceptor molecules to determine binding curves, which bear information about maximal FRET efficiencies and relative binding constants for individual FRET pairings. Calibration factors that represent the optical properties of the imaging setup and the fluorophores are crucial for quantitative measurements. A detailed description how to assess these factors is provided. The quantitative FRET approach is very robust as both donor-centric (E-FRET) and acceptor-centric (SE-FRET) efficiencies are obtained simultaneously from multiple cells. The method was further applied to investigate protein-protein interactions of membrane proteins. First of all, in manuscript II, an epilepsy-causing mutation in the Kv7.2 potassium channel was shown to be implicated in a reduced calmodulin binding affinity to the channel, which affects channel regulation. A second study identified SNARE proteins, such as syntaxin 7 and syntaxin 6, as novel interaction partners of the intracellular ion channel TPC2 (manuscript III), revealing TPC2 as a putative member of the late endosome-lysosome fusion machinery. In manuscript IV, the impact of polymorphic variants of TPC2 on channel dimerization and mTOR binding was investigated. Furthermore, in a study covered by manuscripts V and VI, rhodopsin as well as S- and M-opsins were identified as novel interaction partners of the retinal protein peripherin 2 in rods and cones, respectively. The binding domain underlying the interaction between peripherin 2 and rhodopsin, could be assigned to the fourth transmembrane domain of peripherin 2. Moreover, it could be demonstrated that disease-associated mutations in peripherin 2 attenuated this particular binding, suggesting differential pathophysiological consequences of disrupted interactions in rods and cones. In manuscript VIII, peripherin 2 and its homolog Rom-1 were shown to have opposing effects on rod outer segment targeting of disease-linked peripherin 2 mutants by evaluating their binding affinities. Peripherin 2 is a scaffold protein exclusively expressed in outer segments of rods and cones. As photoreceptors are polarized cells, FRET measurements were not only performed on transfected HEK293 cells but also on acutely isolated outer segments of virally transduced murine photoreceptors (manuscript VII). The results gained in this thesis demonstrate that protein interactions play a crucial role in the regulation of proper protein function. Loss of binding partners or a reduced binding affinity to particular proteins may result in pathophysiological conditions. A deeper knowledge about molecular interactions will contribute to the understanding of cellular mechanisms, etiology of diseases and may further evaluate putative targets of pharmacological interest

A quantitative FRET approach to characterize protein-protein interactions in living cells

The ability of proteins to specifically interact with each other is a key feature in the regulation of biological processes. Knowledge about interaction partners and characterization of protein-protein interactions contribute to the understanding of proper protein function and cell physiology. In particular, Förster resonance energy transfer (FRET) is a suitable method to analyze interactions between proteins in living cells. During the progress of this thesis, a quantitative FRET approach was established that aims to evaluate binding curves for interaction partners. Moreover, the quantitative FRET approach was applied to study biological questions, including the investigation of putative interaction partners of the endolysosomal ion channel two-pore channel 2 (TPC2), the Kv7.2 potassium channel and the photoreceptor-specific transmembrane protein peripherin 2. The FRET approach described in manuscript I computes calibrated FRET efficiencies from fluorescent measurements using three-filter cubes and correlates the FRET efficiencies to the concentration of donor and acceptor molecules to determine binding curves, which bear information about maximal FRET efficiencies and relative binding constants for individual FRET pairings. Calibration factors that represent the optical properties of the imaging setup and the fluorophores are crucial for quantitative measurements. A detailed description how to assess these factors is provided. The quantitative FRET approach is very robust as both donor-centric (E-FRET) and acceptor-centric (SE-FRET) efficiencies are obtained simultaneously from multiple cells. The method was further applied to investigate protein-protein interactions of membrane proteins. First of all, in manuscript II, an epilepsy-causing mutation in the Kv7.2 potassium channel was shown to be implicated in a reduced calmodulin binding affinity to the channel, which affects channel regulation. A second study identified SNARE proteins, such as syntaxin 7 and syntaxin 6, as novel interaction partners of the intracellular ion channel TPC2 (manuscript III), revealing TPC2 as a putative member of the late endosome-lysosome fusion machinery. In manuscript IV, the impact of polymorphic variants of TPC2 on channel dimerization and mTOR binding was investigated. Furthermore, in a study covered by manuscripts V and VI, rhodopsin as well as S- and M-opsins were identified as novel interaction partners of the retinal protein peripherin 2 in rods and cones, respectively. The binding domain underlying the interaction between peripherin 2 and rhodopsin, could be assigned to the fourth transmembrane domain of peripherin 2. Moreover, it could be demonstrated that disease-associated mutations in peripherin 2 attenuated this particular binding, suggesting differential pathophysiological consequences of disrupted interactions in rods and cones. In manuscript VIII, peripherin 2 and its homolog Rom-1 were shown to have opposing effects on rod outer segment targeting of disease-linked peripherin 2 mutants by evaluating their binding affinities. Peripherin 2 is a scaffold protein exclusively expressed in outer segments of rods and cones. As photoreceptors are polarized cells, FRET measurements were not only performed on transfected HEK293 cells but also on acutely isolated outer segments of virally transduced murine photoreceptors (manuscript VII). The results gained in this thesis demonstrate that protein interactions play a crucial role in the regulation of proper protein function. Loss of binding partners or a reduced binding affinity to particular proteins may result in pathophysiological conditions. A deeper knowledge about molecular interactions will contribute to the understanding of cellular mechanisms, etiology of diseases and may further evaluate putative targets of pharmacological interest

AAV Vectors for FRET-Based Analysis of Protein-Protein Interactions in Photoreceptor Outer Segments

Fluorescence resonance energy transfer (FRET) is a powerful method for the detection and quantification of stationary and dynamic protein-protein interactions. Technical limitations have hampered systematic in vivo FRET experiments to study protein-protein interactions in their native environment. Here, we describe a rapid and robust protocol that combines adeno-associated virus (AAV) vector-mediated in vivo delivery of genetically encoded FRET partners with ex vivo FRET measurements. The method was established on acutely isolated outer segments of murine rod and cone photoreceptors and relies on the high co-transduction efficiency of retinal photoreceptors by co-delivered AAV vectors. The procedure can be used for the systematic analysis of protein-protein interactions of wild type or mutant outer segment proteins in their native environment. Conclusively, our protocol can help to characterize the physiological and pathophysiological relevance of photoreceptor specific proteins and, in principle, should also be transferable to other cell types

Peripherin-2 and Rom-1 have opposing effects on rod outer segment targeting of retinitis pigmentosa-linked peripherin-2 mutants

Mutations in the photoreceptor outer segment (OS) specific peripherin-2 lead to autosomal dominant retinitis pigmentosa (adRP). By contrast, mutations in the peripherin-2 homolog Rom-1 cause digenic RP in combination with certain heterozygous mutations in peripherin-2. The mechanisms underlying the differential role of peripherin-2 and Rom-1 in RP pathophysiology remained elusive so far. Here, focusing on two adRP-linked peripherin-2 mutants, P210L and C214S, we analyzed the binding characteristics, protein assembly, and rod OS targeting of wild type (per(WT)), mutant peripherin-2 (per(MT)), or Rom-1 complexes, which can be formed in patients heterozygous for peripherin-2 mutations. Both mutants are misfolded and lead to decreased binding to per(WT) and Rom-1. Furthermore, both mutants are preferentially forming non-covalent per(MT)-per(MT), per(WT)-per(MT), and Rom-1-per(MT) dimers. However, only per(WT)-per(MT), but not per(MT)-per(MT) or Rom-1-per(MT) complexes could be targeted to murine rod OS. Our study provides first evidence that non-covalent per(WT)-per(MT) dimers can be targeted to rod OS. Finally, our study unravels unexpected opposing roles of per(WT) and Rom-1 in rod OS targeting of adRP-linked peripherin-2 mutants and suggests a new treatment strategy for the affected individuals

A modern look at a medieval bilayer metal leaf: nanotomography of Zwischgold

Many European sculptures and altarpieces from the Middle Ages were decorated with Zwischgold, a bilayer metal leaf with an ultra-thin gold face backed by silver. Zwischgold corrodes quickly when exposed to air, causing the surface of the artefact to darken and lose gloss. The conservation of such Zwischgold applied artefacts has been an obstinate problem. We have acquired quantitative, 3D nanoscale images of Zwischgold samples from 15th century artefacts and modern materials using ptychographic X-ray computed tomography (PXCT), a recently developed coherent diffractive imaging technique, to investigate the leaf structure and chemical state of Zwischgold. The measurements clearly demonstrate decreasing density (increasing porosity) of the leaf materials and their corrosion products, as well as delamination of the leaves from their substrate. Each of these effects speak to typically observed issues in the conservation of such Zwischgold applied artefacts. Further, a rare variant of Zwischgold that contains extremely thin multiple gold layers and an overlapping phenomenon of Zwischgold with other metal leaves are observed through PXCT. As supportive data, scanning electron microscopy (SEM) and scanning transmission electron microscopy (STEM) coupled with energy dispersive X-ray analysis (EDX) were performed on the medieval samples

PLOS One / Comparison of EEG-Features and classification methods for motor imagery in patients with disorders of consciousness

Current research aims at identifying voluntary brain activation in patients who are behaviorally diagnosed as being unconscious, but are able to perform commands by modulating their brain activity patterns. This involves machine learning techniques and feature extraction methods such as applied in brain computer interfaces. In this study, we try to answer the question if features/classification methods which show advantages in healthy participants are also accurate when applied to data of patients with disorders of consciousness. A sample of healthy participants (N = 22), patients in a minimally conscious state (MCS; N = 5), and with unresponsive wakefulness syndrome (UWS; N = 9) was examined with a motor imagery task which involved imagery of moving both hands and an instruction to hold both hands firm. We extracted a set of 20 features from the electroencephalogram and used linear discriminant analysis, k-nearest neighbor classification, and support vector machines (SVM) as classification methods. In healthy participants, the best classification accuracies were seen with coherences (mean = .79; range = .53.94) and power spectra (mean = .69; range = .40.85). The coherence patterns in healthy participants did not match the expectation of central modulated -rhythm. Instead, coherence involved mainly frontal regions. In healthy participants, the best classification tool was SVM. Five patients had at least one feature-classifier outcome with p0.05 (none of which were coherence or power spectra), though none remained significant after false-discovery rate correction for multiple comparisons. The present work suggests the use of coherences in patients with disorders of consciousness because they show high reliability among healthy subjects and patient groups. However, feature extraction and classification is a challenging task in unresponsive patients because there is no ground truth to validate the results

CO2Image retrieval studies and performance analysis

Current and planned satellite missions such as the Japanese GOSAT (Greenhouse Gases Observing Satellite) and NASA's OCO (Orbiting Carbon Observatory) series and the upcoming Copernicus Carbon Dioxide Monitoring (CO2M) mission aim to constrain national and regional-scale emissions down to scales of urban agglomerations and large point sources. The CO2Image demonstrator mission of the German Aerospace Center (DLR) is specifically designed to detect and quantify carbon dioxide (CO2) and methane (CH4) emissions from medium-size point sources. To this end its COSIS (Carbon dioxide Sensing Imaging Spectrometer) push-broom grating spectrometer measures reflected solar radiation with a high spatial resolution of 50x50 m2, covering tiles of ~50x50 km2 extent. The instrument has a moderate spectral resolution of approximately ~1 nm and observes in a single spectral window in the 2 µm region. Here we present and discuss the impact of the expected COSIS performance on the retrieved level-2 data. The level-1 data (spectra) are generated using the Py4CAtS (Python for Computational ATmospheric Spectroscopy) line-by-line radiative transfer model and the COSIS SIMulator (COSIS-SIM). Based on the COSIS instrument parameters the analysis examines the retrieval errors related to noise which allows to estimate the detection and quantification limit of CO2 and CH4 emission rates at the instrument's spatial and spectral resolution. We further discuss the effect of heterogeneous scenes, i.e. high contrast surfaces that cause an effective distortion of the spectral response function by non-uniform illumination of the entrance slit. Finally, we assess the influence of initial guess values for the plume's vertical extent and shape on the retrieval

The CO2Image mission: retrieval studies and performance analysis

The CO2Image satellite mission, led by the German Aerospace Center (DLR), aims to demonstrate the feasibility of quantifying carbon dioxide (CO2) and methane (CH4) emissions from medium-size point sources. Several DLR institutes are currently working on the reliminary design phase (Phase B) of the mission. Here we present a performance analysis based on the current instrument specifications. The Beer InfraRed Retrieval Algorithm (BIRRA), the line-by-line radiative transfer model Py4CAtS (Python for Computational ATmospheric Spectroscopy) and a COSIS (Carbon dioxide Sensing Imaging Spectrometer) instrument model are employed to infer CO2 and CH4 concentrations from synthetic COSIS spectra. We evaluate the instrument's performance and determine if it meets the intended requirements. The study assesses uncertainties in the retrieved concentrations as well as errors in point source emission estimates caused by instrument noise. First results suggest that the detection and quantification limits stated in the mission requirements document are justified. The analysis also demonstrates that retrieval errors tend to increase when the signal-to-noise ratio is low, complicating the distinction between emission sources and background concentrations. Furthermore, we discuss non-instrumental effects and demonstrate that the fit quality significantly improves if a low-level plume is scaled instead of a background reference profile that covers the atmosphere's full vertical extent. The analysis on heterogeneous scenes (high albedo contrast) reveals that the various instrument setups perform similarly for both molecules

High susceptibility to fatty liver disease in two-pore channel 2-deficient mice

Endolysosomal organelles play a key role in trafficking, breakdown and receptor-mediated recycling of different macromolecules such as low-density lipoprotein (LDL)-cholesterol, epithelial growth factor (EGF) or transferrin. Here we examine the role of two-pore channel (TPC) 2, an endolysosomal cation channel, in these processes. Embryonic mouse fibroblasts and hepatocytes lacking TPC2 display a profound impairment of LDL-cholesterol and EGF/EGF-receptor trafficking. Mechanistically, both defects can be attributed to a dysfunction of the endolysosomal degradation pathway most likely on the level of late endosome to lysosome fusion. Importantly, endolysosomal acidification or lysosomal enzyme function are normal in TPC2-deficient cells. TPC2-deficient mice are highly susceptible to hepatic cholesterol overload and liver damage consistent with non-alcoholic fatty liver hepatitis. These findings indicate reduced metabolic reserve of hepatic cholesterol handling. Our results suggest that TPC2 plays a crucial role in trafficking in the endolysosomal degradation pathway and, thus, is potentially involved in the homoeostatic control of many macromolecules and cell metabolites

Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice

Lung emphysema and chronic bronchitis are the two most common causes of chronic obstructive pulmonary disease. Excess macrophage elastase MMP-12, which is predominantly secreted from alveolar macrophages, is known to mediate the development of lung injury and emphysema. Here, we discovered the endolysosomal cation channel mucolipin 3 (TRPML3) as a regulator of MMP-12 reuptake from broncho-alveolar fluid, driving in two independently generated Trpml3-/- mouse models enlarged lung injury, which is further exacerbated after elastase or tobacco smoke treatment. Mechanistically, using a Trpml3IRES-Cre/eR26-τGFP reporter mouse model, transcriptomics, and endolysosomal patch-clamp experiments, we show that in the lung TRPML3 is almost exclusively expressed in alveolar macrophages, where its loss leads to defects in early endosomal trafficking and endocytosis of MMP-12. Our findings suggest that TRPML3 represents a key regulator of MMP-12 clearance by alveolar macrophages and may serve as therapeutic target for emphysema and chronic obstructive pulmonary disease